The present invention relates to methods of targeted expression by the cartilage-derived retinoic acid-sensitive protein (CD-RAP) promoter. It is also directed to nucleic acid constructs and vectors useful for expression of nucleic acid sequences targeted to cartilage during development or in adult cartilage tissue, or for their expression in specific tumor cells such as chondrosarcomas and mammary tumor cells. The invention also relates to transgenic mice and other animals altered to contain the CD-RAP promoter transgene.
Cartilage-derived retinoic acid-sensitive protein (CD-RAP) was identified from bovine chondrocytes by the techniques of reverse transcription-polymerase chain reaction (PCR) and differential display during the attempt to identify the molecular markers in the regulation of chondrogenesis (Dietz and Sandell, 1996). The protein was previously isolated and cloned as melanoma inhibitory activity (MIA) from a human melanoma cell line (Blesch et al., 1994). The entire gene encoding CD-RAP/MIA is well conserved among different species, including four exons which are interrupted by three introns (Dietz and Sandell, 1996; Bosserhoff et ,al., 1997a). Functional analysis of the CD-PAP/MIA promoter shows that this gene is actively expressed in the human and murine melanoma cells and its activity is inducible by phorbol ester (Bosserhoff et al., 1996). Unlike other cartilage genes such as collagen types II, IX, and XI, which are also observed in a variety of non-cartilaginous tissues during embryogenesis (Sandell et al., 1991; Cheah et al., 1991; Sandell et al., 1994; Yoshioka et al., 1995), the normal expression of CD-RAP/MIA is restricted to cartilage cells of the chondrocyte lineage and mature chondrocytes. With a more restricted expression pattern in normal tissues, CD-RAP has also proven to be a good model for the studies of the transcriptional regulation of cartilage specific genes. CD-RAP/MIA is activated during the beginning of chondrogenesis and expressed throughout cartilage development (Dietz and Sandell, 1996; Bosserhoff et al., 1997a). One exception to the exclusive expression in cartilage is the expression in premammary buds of the embryo.
So far, little is known about the function of CD-RAP/MIA in chondrogenesis. CD-RAP is coregulated by RA with type II collagen which is the most abundant extracellular protein made by chondrocytes and generally considered to be characteristic of cartilage, suggesting the potential role of CD-RAP in morphogenesis and differentiation of chondrocytes. RA is a potent morphogen in embryogenesis and fetal development and is involved in cartilage differentiation. Applicant""s analysis of the CD-RAP transgene reveals that the CD-RAP expression is correlated with chondrogenesis. But its expression level is decreased in hypertrophic chondrocytes, implying that CD-RAP may be required for the maintenance of chondrocyte phenotype.
The function of CD-RAP/MIA in carcinogenesis and metastasis is also poorly understood. CD-RAP/MIA is detected at various levels in malignant melanoma, chondrosarcoma and mammary carcinomas (Blesch et al., 1994; van Groningen et al., 1995; Bosserhoff et al., 1997a and b; Lu et al., 1997). In primary chondrocytes, CD-RAP/MIA inhibits DNA synthesis (Kondo et al., 1998). Similarly, treatment of a melanoma cell line HTZ-19 with MIA resulted in inhibition of DNA synthesis and significant change of cell morphology as melanoma cells rounded up (Blesch et al., 1994). Evidence suggests that CD-RAP/MIA plays an important role in carcinogenesis and tumor metastasis as CD-RAP/MIA mediates the detachment of melanoma cells from extracellular matrix molecules (Bosserhoff et al., 1997c). Increased serum levels of CD-RAP/MIA were detected in 100% of the patients with metastatic melanoma. In contrast, none of the patients with negative CD-RAP/MIA values developed metastasis, suggesting that CD-RAP/MIA may participate in tumor invasion and metastasis (Bosserhoff et al., 1997b). The CD-RAP/MIA serum level is currently used for the staging and monitoring of metastatic melanoma (Bosserhoff et al., 1997b). CD-RAP/MIA serves as a novel serum marker for malignant melanoma with the highest sensitivity and specificity compared with S-100 and soluble intercellular adhesion molecule 1.
Analysis of CD-RAP transgenic mice demonstrates that CD-RAP is only expressed in mammary gland at very early stage of the development of mammary buds. But it is reexpressed in the chemically induced rat mammary carcinomas. Furthermore, sequence comparison of CD-RAP/MIA gene from malignant tumors with that from normal tissues reveals no mutation in the coding region (Lu et al., 1997). These results support the hypothesis that overexpression of CD-RAP/MIA rather than specific gene mutation may contribute to the carcinogenesis of breast cancer (Lu et al., 1997).
To better understand the functioning of genes, researchers have focused on transcription regulation, including the influence of transcription factors on promoters. Transcription factors, AP-2 and SOX9, have been shown to be important for chondrocyte differentiation (Foster et al., 1994; Wright et al., 1995; Schorle et al., 1996; Zhang et al., 1996; Ng et al., 1997; Zhao et al., 1997). CD-RAP is the first cartilage gene shown to be regulated by AP-2 (Xie et al., 1998). The importance of AP2 in chondrogenesis is demonstrated in AP-2 knock out mice which reveal severe skeletal deformation (Schorle et al., 1996; Zhang et al., 1996). Applicants have shown that AP-2 is biphasically involved in the regulation of CD-RAP promoter activity and response to retinoic acid (RA), specifically, applicants have demonstrated that transactivation by AP-2 contributes to the constitutively high expression of CD-RAP in chondrocytes and that the overexpression of AP-2 induced by RA results in significant reduction of the CD-RAP transcript (Xie et al., 1998).
In addition, SOX9 is able to activate the expression of several cartilage genes, including collagen types II and XI (Lefebvre et al., 1997; Bell et al., 1997; Zhou et al., 1998; Bridgewater et al., 1998). SOX9 protein contains a high mobility group (HMG)-type DNA binding domain and a transactivation domain which was mapped within the 100 C-terminal amino acids (Grosschedl et al., 1994; Sufdbeck et al., 1996). Haploinsufficiency of SOX9 has been attributed to the campomelic dysplasia, a skeletal malformation syndrome (Foster et al., 1994). SOX9 can bind to the CD-RAP promoter and enhances the CD-RAP expression. However, it appears that additional transcription factors are essential for the activation of chondrocyte differentiation, since neither SOX9 nor AP-2 is able to stimulate cartilage gene expression in nonchondrogenic cells (Lefebvre and de Crombrugghe, 1998; Xie and Sandell, unpublished data).
Because CD-RAP may play critical roles in chondrogenesis, carcinogenesis, and metastasis, deeper understanding of its transcriptional regulation, especially in specific tissues, can lead to effective design of therapeutic agents and diagnostics and research tools. Tissue specific gene expression in cartilage, as well as in tumor cells such as chondrosarcoma or mammary tumors is frequently desirable. For example, growth factors or enzymes could be targeted to cartilage tissue to enhance tissue regeneration or repair without affecting surrounding tissue. Similarly, drugs designed to inhibit tumor cell growth or antigenic proteins which can be used to trigger an immune response could thereby be selectively delivered to the tumor cells with minimal adverse impact on surrounding healthy tissue.
Among the objects of the invention, therefore, may be noted the provision of methods of targeting expression of desired nucleic acid sequences specifically to cartilage primordial cells and adult articular cartilage cells. Also provided is a means for expressing desired nucleic acid sequences specifically in chondrosarcoma or mammary tumor cells. Nucleic acid constructs and vectors for the introduction of the desired nucleic acid sequences and transgenic animals altered to express desired nucleic acid sequences in the above-mentioned specific tissue cells are also provided.
Thus, the present invention is directed to a method for the expression of a desired nucleic acid sequence in cartilage cells of the chondrocyte lineage or mature chondrocyte cells comprising the steps of transfecting the cells with a nucleotide construct containing a murine CD-RAP gene 5xe2x80x2-flanking segment capable of targeting such cells for expression operably linked to a nucleic acid sequence to be expressed and expressing the desired nucleic acid sequence. Methods employing these steps for targeted expression in chondrosarcoma or mammary tumor cells or cell lines derived therefrom are also provided.
The invention is further directed to a nucleotide construct comprising a murine CD-RAP gene 5xe2x80x2-flanking segment operably linked to a foreign nucleic acid sequence.
A further aspect of the present invention is directed to a nucleotide construct comprising a human CD-RAP gene 5xe2x80x2-flanking segment operably linked to a foreign nucleic acid sequence.
The invention also relates to application of a recombinant transcription factor, such as sox9, to alter expression of a CD-RAP promoter construct.
The present invention is also directed to a vector for expression of a desired nucleic acid sequence comprising a nucleotide construct containing a murine CD-RAP gene 5xe2x80x2-flanking segment and said nucleic acid sequence wherein said nucleic acid sequence is located in sequential and positional relationship for expression of the nucleic acid sequence.
A further aspect of the present invention is directed to a vector for expression of a desired nucleic acid sequence comprising a nucleotide construct containing a human CD-RAP gene 5xe2x80x2-flanking segment, a reporter sequence, and said nucleic acid sequence wherein said nucleic acid sequence is located in sequential and positional relationship for expression of the nucleic acid sequence.
The invention is additionally directed to a transgenic animal comprising such a nucleotide construct.